Calcium dipicolinate (CaDPA, calcium 2,6-pyridinedicarboxilate) represents 5 to 15% by weight of Bacillus anthracis spores (Murrell, W G, in The Bacterial Spores, Gould, G W, A Hurst, Eds. Academic Press, London, 1969, p. 215), and consequently a number of methods are being developed and used to detect CaDPA or its derivatives as an indicator or biochemical signature for the presence of B. anthracis, a bacterium that has been used in bioterrorism due to its ability to cause anthrax. The appropriateness of measuring CaDPA or its derivatives as a signature for B. anthracis is supported by the fact that only spore-forming bacteria contain CaDPA, of which there are 13 genera, two of which, Bacillus and Clostridium, are common and of interest; while the most widespread, potentially interfering spores, such as pollen and mold spores, do not contain CaDPA. This ability to discriminately detect anthrax-causing spores is one of the important measurement parameters that must be satisfied if a method is to have value in minimizing terror. Three additional germane measurement parameters include sensitivity, speed and ease-of-use. The Center for Disease Control estimates that inhalation of 10,000 spores, or 100 nanograms, will be lethal to 50 percent of an exposed population (Ingelsby, T V et al., “Anthrax as a biological weapon, 2002: Updated recommendations for management” J Amer Med Ass, 287, 2236-52, 2002). The ideal measurement device would have as a minimum sensitivity the ability to detect the presence of 10,000 spores within minutes of detection, such that precautions could be executed to prevent infection. Such a device could be used to detect spores in suspicious mail or on contaminated surfaces, and prevent distribution or exposure.
Methods that have successfully been used to detect CaDPA or its derivatives include mass spectrometry and fluorescence, luminescence, and Raman spectroscopies. Mass spectrometry has been used to identify dipicolinic acid (DPA), the acid form of CaDPA, that was separated from spores by pyrolysis (Beverly M B, F Basile, K J Voorhees, T L Hadfield, “A rapid approach for the detection of dipicolinic acid in bacterial spores using pyrolysis/mass spectrometry”, Rapid Commun. Mass Spectrom, 10, 455-458, 1996). Although this method provides a relatively high degree of discrimination and sensitivity, it requires significant time due to sample handling and data analysis.
Fluorescence involves of course the absorption of electromagnetic radiation into an electronic transition of a molecule and the re-emission of radiation at longer wavelengths. It has been shown that fluorescence can be obtained from CaDPA (Nudelman, R, B V Bronk, S Efrima, “Fluorescence Emission Derived from Dipicolinate Acid, its Sodium, and its Calcium Salts” Appl Spectrosc 54, 445-449, 2000), however the emission spectrum is not sufficiently unique to differentiate it from common biological materials that also fluoresce. In an effort to overcome this limitation, others have explored the formation and the use of a terbium-DPA complex to generate a distinctive, highly luminescence spectrum (here defined as fluorescence that employs a chemical modification, Rosen D L, C Sharpless, L B McBrown, “Bacterial spore detection and determination by use of terbium dipicolinate photoluminescence”, Anal Chem, 69, 1082-1085, 1997; or Rosen D L et al. U.S. Pat. No. 5,876,960). In a similar study, hot dodecylamine (DDA, a cationic surfactant) was used to rapidly break apart the spore to release CaDPA, as the acid, to form the terbium complex (Pellegrino P M, N F Fell Jr., J B Gillespie, “Enhanced spore detection using dipicolinate extraction techniques” Analyt Chim Acta, 455, 167-177, 2002). Unfortunately, it has been found that as many as three concentration-dependent complexes can form, each with different lifetimes (Rosen D L, S Niles, “Chelation Number of Terbium Dipicolinate: Effects on Photoluminescence Lifetime and Intensity”, Appl Spectrosc 55, 208-216, 2001). This, coupled with the fact that the Tb3+ cation produces the same luminescence spectrum, makes determinations of low spore concentrations problematic. Furthermore, the combination of heat and the DDA surfactant severely degrade the spore, generating cell debris (here defined as biochemical fragments of the spore). This requires sample cleanup and in this particular case, AlCl3 had to be added to remove phosphates that would interfere with the photoluminescent measurement.
It has long been known that Raman spectra of bacilli spores are dominated by peaks associated with CaDPA, and that these spectra may provide a suitable anthrax signature at the genus level (Woodruff W H, T G Spiro, C Gilvarg, “Raman Spectroscopy In Vivo: Evidence on the Structure of Dipicolinate in Intact Spores of Bacillus Megaterium”, Biochem Biophys Res Commun, 58, 197-203, 1974). Since that time, considerable improvements in Raman instrumentation have led to field measurements of spores captured from a mail-sorting system (Farquharson S, L Grigely, V Khitrov, W W Smith, J F Sperry, G Fenerty, “Detecting Bacillus cereus spores on a mail sorting system using Raman Spectroscopy”, J Raman Spectrosc, 35, 82-86, 2004). However, Raman spectroscopy is inherently an insensitive technique, and these measurements were of milligram, and not the required nanogram, sample amount.
Two approaches are widely used to improve the sensitivity of Raman spectroscopy, i.e., resonance Raman spectroscopy and surface-enhanced Raman spectroscopy (SERS). The former method involves laser excitation at or near the wavelength of an electronic absorption to substantially increase the interactions between the radiation and molecular states, and was used more than a decade ago to analyze Bacillus spores (Ghiamati E, R S Manoharan, W H Nelson, J F Sperry, “UV Resonance Raman spectra of Bacillus spores”, Appl Spectrosc, 46, 357-364, 1992). The value of this technique is limited by the extremely low energy conversion of ultraviolet lasers, which require substantial power supplies and thus confine measurements to laboratory settings, and which also require spectral acquisition times as long as one hour.
SERS involves the absorption of incident laser photons within nanoscale metal structures, generating surface plasmons, which couple with nearby molecules (the analyte) and thereby enhance the efficiency of Raman scattering by six orders of magnitude or more (Jeanmaire D L, R P Van Duyne, “Surface Raman Spectroelectrochemistry”, J Electroanal Chem, 84, 1-20, 1977; or Weaver M J, S Farquharson, M A Tadayyoni, “Surface-enhancement factors for Raman scattering at silver electrodes: Role of adsorbate-surface interactions and electrode structure”, J Chem Phys, 82, 4867-4874, 1985). In addition to affording high levels of sensitivity, the rich molecular vibrational information provided by Raman scattering yields exceptional selectivity and allows the identification of virtually any chemical as well as the ability to distinguish multiple chemicals in mixtures (see Garrel R L, “Surface-Enhanced Raman Spectroscopy”, Anal Chem, 61, 401A-411A, 1989; or Storey J M E, T E Barber, R D Shelton, E A Wachter, K T Carron, Y Jiang, “Applications of Surface-Enhanced Raman Scattering (SERS) to Chemical Detection”, Spectroscopy, 10, 20-25, 1995).
Four methods have become common in the practice of generating SERS. They are: (1) activated electrodes in electrolytic cells (see for example Jeanmaire or Weaver above); (2) activated silver and gold colloids (Kerker M, O Siiman, L A Bumm, D-S Wang, “Surface-enhanced Raman Scattering of citrate ion adsorbed on colloidal silver”, Appl Opt, 19, 3253-3255, 1980, or Angel S M, L F Katz, D D Archibold, L T Lin, D E Honigs, “Near Infrared Surface-enhanced Raman Spectroscopy. Part II: Copper and gold colloids”, Appl Spectrosc, 43, 367-372, 1989); (3) activated silver and gold substrates (Seki H, “Surface-enhanced Raman Scattering of pyridine on different silver surfaces”, J Chem Phys, 76, 4412-4418, 1982, or Li Y-S, T Vo-Dinh, D L Stokes, Y Wang, “Surface-Enhanced Raman Analysis of p-Nitroaniline on Vacuum Evaporation and Chemical Deposited Silver-Coated Alumina Substrates”, Appl Spectrosc, 46, 1354-1357, 1992); and (4) sol-gels doped with silver or gold particles (Farquharson et al. U.S. Pat. Nos. 6,623,977, 6,943,031, and 6,943,032 and corresponding International Application Publication No. WO 01/33189 A2, which are commonly owned herewith and the entire specification of which the United States patents are hereby incorporated by reference thereto).
The first measurement of dipicolinic acid by SERS was reported in 1999 followed by a more in-depth analysis in 2004 (Farquharson S, W W Smith, S Elliott, J F Sperry, “Rapid biological agent identification by surface-enhanced Raman spectroscopy”, SPIE, 3855, 110-116, 1999; Farquharson S, A Gift, P Maksymiuk, F Inscore, and W Smith, “pH dependence of methyl phosphonic acid, dipicolinic acid, and cyanide by surface-enhanced Raman spectroscopy”, SPIE, 5269, 117-125, 2004). In these two reports, the measurement of DPA by SERS was performed to demonstrate the possible use of such a measurement to identify spores, such as Bacillus anthracis. However, both measurements were of pure DPA in water and no method to extract the DPA from spores was mentioned. In any event, no prior art describes the use of chemicals to cause the release of CaDPA so that it or DPA is available for measurement by SERS; that concept is disclosed in recent papers authored by the present inventors (see Farquharson S, A Gift, P Maksymiuk, F E Inscore, “Rapid dipicolinic acid extraction from Bacillus spores detected by surface-enhanced Raman spectroscopy”, Appl Spectrosc, 58, 351-54, 2004; and Inscore F E, A D Gift, S Farquharson, “Detect-to-treat: development of analysis of Bacilli spores in nasal mucus by surfaced-enhanced Raman spectroscopy”, SPIE, 5585, 53-57, 2005).
Since the Farquharson, et al. publication (Appl Spectrosc, 2004) one research group has attempted to improve upon these measurements (Zhang X, M A Young, O Lyandres, R P Van Duyne, “Rapid Detection of an Anthrax Biomarker by Surface-Enhanced Raman Spectroscopy”, J Am Chem Soc, 127, 4484-4489, 2005). In this paper, published on Mar. 30, 2005, 0.02M nitric acid was used in conjunction with 10 minutes of ultrasonification to obtain a SER spectrum of DPA. Although a 200 fold improvement in sensitivity was claimed, the lowest reported amount measured, 187,000 spores per microliter, was considerably less sensitive than the 10,000 spores per microliter reported by Farquharson et al. Xhang et al. also reported that no signal was obtained unless sonication was used.
Even so, in these measurements, the use of DDA or nitric acid alone was insufficient to separate a significant amount of DPA (defined, for present purposes, to be at least 10% by weight, on average, of that which is available) in a timely manner (defined, for present purposes, as a period that is less than 30 minutes, and preferably less than 10 minutes), without the addition of heat or ultrasound. The use of heat or ultrasound had two deleterious consequences, 1) it produced spore debris that likely reduced the sensitivity of the SERS measurement, and 2) it unduly complicated the device by requiring a heat or ultrasound source.
Significant research to understand the sporulation and corresponding germination of spores has been performed for more than 50 years. Bacterial spores are considered marvels of nature, in that when conditions for life become unfavorable, they become dormant through a process known as sporulation (forming an endospore). These spores are resistant to severe environmental conditions, such as heat, ultraviolet radiation, vacuum, and many chemicals including oxidizing agents and strong acids (e.g. nitric, hydrochloric, sulfuric, etc., see Russell, A D, The destruction of bacterial spores, Acad Press, New York, N.Y., 1982 or Nicholson, et al. “Resistance of Bacillus Endospores to Extreme Terrestrial and Extraterrestrial Environments”, MicroBio Molec Bio Rev, 64, 584-572, 2000). However, when conditions are again favorable they re-initiate cell growth through a process known as germination (see Dricks, A, “From Rings to Layers: Surprising patterns of protein deposition during bacterial spore assembly”, J Bacteriol, 186, 4423-4426, 2004). Numerous methods have been examined to initiate germination to elucidate this process. Germinants studied naturally included amino and nucleic acids, sugars, carbohydrates, and also dodecylamine (Setlow B, A E Cowan, P Setlow, “Germination of spores of Bacillus subtilis with dodecylamine”, J Appl Microbiol, 95, 637-648, 2003). It is now known that certain amino acids and inosine, a nucleic acid variant, initiate germination, which includes an early step involving the release of CaDPA, which becomes DPA as it enters the surrounding solution. It is also now known that DDA initiates germination through a different mechanism. The details of these mechanisms whereby CaDPA is released using natural nutrients or DDA, through one or multiple spore wall channels, are still unknown. In fact, endospores may contain a small percent of DPA along with the CaDPA. In either case, however, germination takes several hours at room temperature, and consequently none of these chemicals provide sufficient speed for the purpose to satisfy the objects of this invention.
In another area of research, methods have been investigated for the purpose of killing spores, such as in the case of sterilizing medical equipment and disinfecting food. A comprehensive review is given by Russel A D, “Bacterial Spores and Chemical Sporicidal Agents”, Clin Microbiol Revs, 3, 99-119, 1990. In summary, only few chemicals kill spores effectively, glutaraldehyde, formaldehyde, chlorine releasing agents, peroxygens (e.g. peroxide), and ethylene oxide, of which only the first two have practical value. It is worth noting that according to this review organic acids, specifically, benzoic acid and sorbic acid, can be used to kill bacterial cells, but not spores. In recent years the mechanisms by which these chemicals, and others, kill spores have been examined. However, only limited studies have involved the analysis of DPA. These reports measure the release of DPA as a method of improving the understanding of germination, as well as spore death. These reports include the use of inorganic acids, alkali, peroxides, and haloginated chemicals (Young S B, P Setlow, “Mechanisms of killing of spores of Bacillus subtilis by iodine, glutaraldehyde and nitrous acid”, J Appl Microbiol, 89, 330-338, 2000, Setlow B, C A Loshen, P C Genest, A E Cowan, C Setlow, P Setlow, “Mechanisms of killing spores of Bacillus subtilis by acid, alkali, and ethanol”, J Appl Microbiol, 92, 362-375, 2002, and Young S B, P Setlow, “Mechanisms of killing of Bacillus subtilis spores by hypochlorite and chlorine dioxide”, J Appl Microbiol, 95, 54-67, 2003). In all of these cases, hours of treatment are required to generate significant quantities of DPA. Again, however, the inclusion of a physical means, such as heat, pressure, sonication, or their combination (e.g. autoclaving), are required if the DPA release times are to be substantially reduced, and in such cases significant cell debris is generated, thus compromising the sensitivity of DPA detection.
It is therefore surprising that treatment of spores by a weak acid compound, having a pKa value of 0.1 to 11, at room temperature, in accordance with the present invention and as is described hereinafter, quickly (e.g., within 10 minutes) releases most, if not all, of the CaDPA contained therein.